The relay of extracellular signals of 3',5' cyclic adenosine monophosphate (cAMP) mediates the highly organized aggregation of single D. discoideum amoebae to form a multicellular structure. Previous investigations have shown that amoebae are attached to cAMP sources and also transiently produce and secrete cAMP in response to exogenous cAMP stimuli. The biochemical mechanisms by which the occupancy of surface cAMP receptors controls the activation of adenylate cyclase will be investigated by measuring rates of secretion of 3H-cAMP by intact cells, rates of 32P-cAMP synthesis by broken cell preparations, and by employing a histochemical stain to determine the subcellular localization of the enzyme. Preliminary observations have shown that NaN3 blocks activation of the adenylate cyclase by extracellular cAMP and that folic acid-related compounds, added to intact cells, can activate the enzyme. In the investigation of the mechanisms which lead to activation of the adenylate cyclase, the biochemical basis of the effects of NaN3 and folic acid will be explored. A selection procedure to isolate mutants in which the adenylate cyclase is persistently (rather than transiently) activated is described. The cAMP waves which guide the coordinated aggregation of D. discoideum amoebae will be studied with a recently discovered solid-phase isotope dilution-fluorography technique. This technique measures the distribution of cAMP on a two-dimensional surface to a resolution of 0.1-0.3 mm. The direct visualization of the cAMP waves in situ will permit a close correlation of observations of group cell behavior and biochemical observations of the cAMP signaling response.